Analysis of Nitrate and Phosphate in St. John’s Lakes and Surrounding Areas
Kristi Strandberg and Dr. Michael Ross
Department of Chemistry
Saint John’s University/ College of Saint Benedict
St. Joseph, MN 56374
Summer, 2002
Abstract
Water samples were collected weekly from several sites surrounding St. John’s
University, St. Joseph, and Sartell. These samples were analyzed to determine their
nitrate and phosphate levels. The primary focus of this research was to determine the
effects of the St. John’s sewage treatment plant on the surrounding ecosystem, and then
to compare this to the effects that local farming and agriculture have on the water
systems in St. Joseph and Sartell. This research found that only two sites were above the
levels recommended by the Environmental Protection Agency (which is 1 ppm for both
nitrate and phosphate1). East Gemini Lake, which is where the treated water is initially
emptied, was slightly above the recommended level. The stream found at Millstream
Park was also slightly above the recommended level.
INTRODUCTION
Originally St. John’s University operated only with a septic system to treat
wastes. However, as the school expanded, the septic system was not capable of handling
the increased amount of wastewater. It was because of this growing problem that the
sewage treatment plant was built at St. John’s. The East Gemini Lake was also created to
help reduce the nitrates and phosphates in the water as it leaves the treatment plant. The
water that leaves the plant usually contains high levels of nitrate and phosphate, which
can be detrimental to the aquatic life found in lakes and their surrounding ecosystems2.
The possibility of eutrophication caused by the overgrowth of algae is of great concern.
Large amounts of algae are directly related to high phosphate and nitrate levels. Both
East Gemini Lake and the St. John’s wetlands were designed to help lower the levels of
nitrate and phosphate. Samples were collected from several sites in an effort to monitor
the nitrate and phosphate levels. The nitrate concentration was analyzed by using
cadmium reduction/ azo-dye using spectroscopy3. The phosphate concentrations were
determined by using UV spectroscopy along with using ascorbic acid and ammonium
molybdate to form the phosphomolybdate complex 4. The results from this research
suggest that East Gemini and the wetlands are still removing the high concentrations of
nitrate and phosphate from the water. Results also indicate that excessive rainfall greatly
increases the nitrate levels. This is possible due to the runoff from fertilizers used by the
local farming communities and by SJU.
EXPERIMENTAL
Water samples were collected from sites around the Collegeville/ St. Joseph area.
These sites are found on the map found in figure 1. The samples were collected at the
same location each time. The samples were also collected on the same day of the week
(sites A-F and H were collected every Monday, and sites G and I-M were collected every
Thursday). The samples were then brought to the laboratory and filtered using glass fiber
filters. The samples were stored in a cooler overnight and the analyzed within 24 hours
of collection.
FIGURE 1
NITRATE METHOD5
Preparation of reagents:
Copper/ Cadmium Granules: 40 g of cadmium granules (0.5 to 2nm in size) were
washed with 2N HCl and then rinsed with distilled water. The granules were then
swirled in 10 mL of 0.008M cupric sulfate solution. The granules were swirled in this
solution until the liquid no longer contained any blue coloring. This solution was then
decanted and a brown precipitate remained on the surface of the cadmium granules.
These granules were rinsed with distilled water and then were ready to be packed in to
the column. The granules were not allowed to come in to contact with the air after being
washed.
Nitrate Buffer: 100 g of ammonium chloride, 20g of sodium tetraborate, and 1g of
disodium dihydrate EDTA was dissolved in distilled water and then diluted to 1liter.
Sulfanilamide solution: 5g of sulfanilamide was dissolved in to 50 mL of
concentrated HCl and 300mL distilled water. This solution was further diluted to
500mL.
N-(1-naphthyl) ethylene diamine dihydrochloride solution: 0.5g of
dihydrochloride was dissolved in 500mL of distilled water. This solution had to be
stored in a brown or amber colored glass bottle. The solution needed to be replaced
about once a month.
Nitrate Standard: Dissolving 0.722g of KNO3 in distilled water and diluting this
to 1L made the original nitrate standard. Prior to dissolving the potassium nitrate, the
compound was dried in an oven for 1h at 110ºC. The nitrate standard contained 100ug of
NO3-N per mL of the standard solution. When preparing the set of standards that were to
be run on the cadmium reduction column, the nitrate standard was diluted by a factor of
100 to obtain a nitrate concentration that would be within the expected range.
Initially the set of standards used included concentrations (in ug/L) of 1.2, 1.8, 3,
6, 12, 18, 30, 60, 120, 180, and 300. It was found that the instruments used were not very
accurate at analyzing concentration that were below 1ug/L. Eventually fewer standards
were needed to accurately analyze the samples. In these situations, only the necessary
standards were prepared and analyzed.
Column Preparation: The column was initially filled with distilled water, and
then the Cd/Cu granules were slowly added. The granules remained under water during
the entire packing process to avoid the formation of air bubbles. After packing, 100mL
of distilled water were drawn through the column. This was followed by 100mL of a
solution that was 50mL buffer and 50mL distilled water. After this the column was ready
for sample analysis. When not in use the column was stored under a diluted nitrate buffer
solution.
The purpose of this column was to allow the nitrate to react with the Cu ions
present on the cadmium granule surface. These Cu ions worked to reduce the nitrate to
nitrite. The hydrochloric acid present in the solution creates an acidic environment for
the reaction to take place in. In such an environment, the nitrite reacts with the
sulfanilamide to form a diazonium compound. Then this compound reacts with the N-(1naphthyl) ethylene diamine dihydrochloride solution. This reaction produces a pink
colored azo-compound.3 When the analysis was performed the level of nitrite was
actually measured instead of nitrate. Previous studies have shown that the lakes
contained a negligible amount of nitrite originally4.
Each sample was measured out to 100mL in a volumetric flask. Then 10mL of
the nitrate buffer solution was added. This sample was thoroughly mixed and then added
to the top of the column. The sample was collected at a rate of about 6mL per 5 minutes.
The samples were collected in 3 x 25mL fractions. The first 15mL of sample to come off
of the column was discarded.
After collecting a fraction, 0.5mL of sulfanilamide solution was added to the
sample and mixed thoroughly. After 5 minutes, but not exceeding 8 minutes 0.5mL of
the naphthyl ethylenediamine dihydrochloride solution was added to the fraction. This
was mixed immediately. After mixing, the sample would develop a pink/ purple
coloration. The intensity of the coloration directly related to the concentration of nitrate
in the water.
The sample was then analyzed with the diode array spectrophotometer, with a
focus on the absorbance at the 542nm wavelength. This analysis needed to be completed
between 10 minutes and 2 hours after adding the dihydrochloride solution.
Calibration: Nitrate standards were made up each time any sample was run on the
column. Each set of standards were used to make a calibration curve (sample calibration
curves are included in later figures).
NITRATE SELECTIVE ELECTRODE6
Ionic Strength Adjuster: This solution is composed of 2M (NH2)SO4.
The samples were measured out it to 10mL portions and placed in a large test
tube. Then to each test tube, four drops of the ionic strength adjuster was added. After
adding the drops, the sample was ready to be tested. Each test tube was placed on the end
of the electrode and then the reading was measured in mV. To plot the results it was
necessary to use the log of the concentration.
PHOSPHATE METHOD1,5
Preparation of reagents
Ammonium Molybdate Solution: 15g of ammonium paramolybdate were
dissolved with distilled water and diluted to 500mL. This solution was supposed to be
stored in an amber colored polyethylene bottle, however, this could not be found so an
amber colored glass bottle was substituted.
Sulfuric Acid Solution: 155mL of concentrated sulfuric acid was diluted to 1L.
Ascorbic Acid Solution: 27g of L-ascorbic acid was dissolved in distilled water
and diluted to 500mL. This solution had to be made daily or frozen in a plastic bottle.
Potassium Antimonyl-tartrate Solution: 0.34g of potassium antimonyl-tartrate was
dissolved in distilled water and diluted to 250mL. The solution was then stored in a glass
bottle.
Composite Reagent: This solution is a mixture of several solutions. It consists of
100mL ammonium molybdate solution, 100mL of the ascorbic acid solution, 50mL of the
potassium antimonyl-tartrate solution, and 250mL of the sulfuric acid solution. The
composite reagent is prepared each time a set of standards/ samples are to be analyzed.
The solution is only good for one day and needs to be discarded after 24hours.
Phosphate Standard: Measuring out 0.2197g of potassium dihydrogen phosphate
and drying it in an oven for 24hours at a temperature of 105ºC prepared the phosphate
standard. After drying, the compound was dissolved in distilled water and diluted to 1L.
The solution was then stored in a dark glass bottle with 1mL of chloroform. The standard
had a 50.0 ug of PO4-P per mL. This standard was further diluted by a factor of 100 to
create a concentration that would be more efficient for preparing the sets of standards.
Initially the set of standards used included concentrations (in ug/L) of 10, 50, 100,
150, 200, and 400. Eventually fewer standards were needed as it became possible to
estimate the approximate range needed for each set of standards.
To analyze the collected samples the composite reagent was prepared and
appropriate standards were made and tested. The samples (which were kept refrigerated
over night) were allowed to sit out until they reached a temperature between 15-30ºC.
These samples were measured out in to 100mL portions using a volumetric flask.
To each portion, 10mL of the composite reagent was added. This was then stirred
vigorously. 10 minutes after adding the composite reagent, the sample was ready for
analysis. Each sample needed to be analyzed between 10 minutes and 2 hours after
adding the composite reagent.
The composite reagent reacted with the phosphates found in the water samples
forming a large phospho-molybdate complex. When this complex is in the presence of
ascorbic acid, the complex is reduced and a blue discoloration can be noticed. The
intensity of the blue coloration reflects how much phosphate is present in the sample. A
Spec 20 Spectrophotometer is then used to measure the absorbance of the sample. The
wavelength the samples were measured at was 885nm. The previously measured
standards are then used to make a calibration curve. From the line of regression on the
calibration curve, the concentration of the samples can be determined.
PHOSPHATE EXTRACTION METHOD5
It was uncertain if the instrument used to perform the analysis was accurately
measuring the absorbencies of low phosphate concentrations, so an alternative method
was attempted.
In this alternative procedure, an organic solvent was used to extract the blue
phospho-molybdate complex. The organic solvent used for this extraction was butyl
acetate( this solvent was chosen based on its availability, even though other solvents such
as isobutanol are more soluble).
In this procedure, 200mL of sample were needed and were measured out using
200mL volumetric flasks. To each 200mL portion of sample, 20mL of the composite
reagent were added and vigorously mixed. Each sample was mixed in a 250mL
separatory funnel. It was then necessary to wait for about 20 minutes for color
development. After 20 minutes, 10mL of butyl acetate was added to each sample by
using a 10mL volumetric pipette. This was shaken vigorously for about 5 minutes.
The organic and aqueous phases were allowed to separate. Then the aqueous
layer was drained off and discarded. The organic layer was retained and analyzed using
the Spec 20. The blank used for this method was pure butyl acetate.
This method was designed to measure lower concentrations of phosphate.
Because it was used to measure lower concentrations, a lower set of standards was
needed. The standards for this method include the concentrations; 1, 5, 10, 15, and
20ug/L. A calibration curve and linear regression were also prepared from this data.
RESULTS
The nitrate and phosphate levels were tested once a week at 13 different sites.
Five of these sites surrounded St. John’s. These included Lake Sagatagan, Stumpf Lake,
East Gemini, the wetlands, and the bridge between East Gemini and Stumpf. The
remaining sites are all along the Watab River and were chosen to monitor the levels of
nitrate and phosphate as the water travels from SJU to the Mississippi.
The results obtained from the nitrate data suggested that the cadmium ion
reduction column was able to repeatedly produce linear calibration lines. Past research
showed that this type of column usually does not produce accurate results or linear
calibration lines. In past studies, the R2 values were of 0.91 or worse and the nitrate
selective ion electrode was much more accurate at measuring the levels of nitrate6.
However, this summer it was found that the cadmium reduction ion was accurate at
reducing the nitrates. The column produced results that had R2 values ranging from 0.970.999. The nitrate electrode was also attempted, but was found to be unable to accurately
measure the nitrate levels (see Figure 2).
FIGURE 2.a
Nitrate Electrode Calibration Curve
250
mV
200
150
Standards
100
50
0
0
0.5
1
1.5
2
Log Concentration
2.5
3
FIGURE 2.b
Nitrate Calibration Curve
1
Absorbance
0.8
0.6
Standards
Regression
0.4
0.2
0
-0.2 0
100
200
300
400
Concentration (ug/L)
Figure 2. These graphs illustrate the differences between the cadmium ion
reduction column and the nitrate electrode. The electrode did not produce results
that were sensitive enough to continue using this method.
The results were recorded in ug/L. The Environmental Protection Agency has set
limits of 225ug/L for nitrate. Based on what is recommended by the EPA, most sites
sampled at were within regulations. However, a few of the sites did exceed the
recommended nitrate level (see Figure 3). East Gemini and part of Stumpf Lake were
above the 225ug/L limit. A couple of points along the Watab River occasionally went
over the limit as well (this seemed to occur mainly after large amounts of rainfall).
FIGURE 3
Nitrate Concentrations
700
600
Concentration (ug/L)
500
6/17-6/21
6/24-6/28
7/8-7/12
7/15-7/19
7/22-7/26
7/29-8/2
400
300
200
100
ip
pi
4
is
s
H
is
s
w
2
H
w
k
Pa
r
ill
St
re
M
C
M
H
am
w
16
0
w
50
H
3
w
H
et
la
nd
ol
le
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vi
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R
d
W
in
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/G
pf
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St
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tG
em
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0
Sample Site
Figure 3. This figure shows the weekly variations in the concentration of nitrate.
225 ug/L was the recommended limit for nitrate. Several sites exceed this limit at
various dates during this summer.
The phosphate samples were collected weekly and analyzed using the Spectronic
20D+ instrument. The results were also recorded in ug/L. 326 ug/L of phosphate is the
recommended limit set by the EPA. East Gemini was repeatedly over this limit, and at
one point during the experiment; Millstream Park was also over the limit. The rest of the
sites maintained levels that were below what was recommended by the EPA (refer to
Figure 4).
FIGURE 4
Phosphate Concentrations
1200
Concentration (ug/L)
1000
800
6/17-6/21
6/24-6/28
7/8-7/12
7/15-7/19
7/22-7/26
7/29-8/2
600
400
200
ip
pi
4
is
s
H
is
s
w
2
H
w
k
Pa
r
ill
St
re
M
C
M
H
am
w
16
0
w
50
H
3
w
H
et
la
nd
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R
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in
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/G
pf
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St
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Sa
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0
Sample Site
Figure 4. This figure illustrates the variations of the phosphate concentrations
throughout the summer. Note the gradual downward slope in the concentration
levels. This suggests that East Gemini and the wetlands are successful in
reducing the phosphate levels before the water reaches the Mississippi.
An additional method for measuring low phosphate concentrations was also
attempted through using an organic solvent to extract the phospho-molybdate complex.
However, this method did not produce results that were accurate enough to replace to
previously chosen method (see Figure 5).
FIGURE 5.a
Absorption
Phosphate Extraction Calibration
Curve
0.03
0.02
0.01
0
-0.01 0
-0.02
Standards
Regression
10
Concentration (ug/L)
20
FIGURE 5.b
Absorbance
Phosphate Calibration Curve
0.4
0.3
0.2
0.1
0
Standards
regression
0
200
400
600
Concentration (ug/L)
Figure 5. This figure illustrates the differences in accuracy between the two
phosphate methods attempted. 5.a demonstrates the irreproducibility of the
results experienced with using the extraction method.
DISCUSSION
The results for the phosphate and nitrate tests have shown a general decrease in
the levels as the water travels down the Watab River towards the Mississippi. The first
site is at Lake Sagatagan which does not flow into the Watab and is used as a
background/reference point. The obtained results show very low concentrations of nitrate
and phosphate. Similar results were noticed for Stumpf Lake. This sample site did not
have any of the treatment plant water emptying into it. This probably accounts for the
low concentrations. Also both of these sites were located away from farms and did not
have nitrates found in fertilizers flowing in to them. These sites did not have the waste
water mixing in with the lake water and increasing the phosphate concentrations.
The bridge between Stumpf and East Gemini had some very high nitrate levels
during a couple of the weeks. This could have been caused by run off from rainstorms.
This area of Stumpf Lake is located on the college campus therefore the nitrate levels
could easily be influenced by the chemicals used to treat the lawns and gardens.
Residential buildings also surround this area of the lake and could also impact the nitrate
levels and cause them to rise. The wastewater did not dump in to this lake, and therefore
did not increase the phosphate levels. This lake had rather low phosphate levels, which
was expected.
Compounds such as phosphate and nitrate are considered nutrients to organisms
such as algae2. East Gemini contains high levels of phosphate and nitrate which both
promote algae growth, however, nitrogen appears to be the limiting factor. As the algae
grow, they use up the nitrate causing the nitrate levels to drop. The nitrate runs out,
resulting in the lower concentrations that were observed during this research.
This same trend can be noticed in the wetlands. The nitrate levels are diminished
by the large quantities of algae. Lots of algae can grow because of high phosphate levels;
however, this growth is eventually inhibited by the decreasing amounts of nitrate (which
is essential for the continued growth of the algae). Also, both East Gemini and the
wetlands have relatively slow moving water. This also makes it easier for the algae to
grow.
After water leaves the wetlands, it continues down the Watab River. The water
moves more rapidly after exiting the wetlands. The increased rate of flow inheres the
growth of algae. The lack of algae and the farms that have fertilizer run off in to the river
around the Old Collegeville Road. This can account for the increase in nitrate. Thje
wetlands and East Gemini were created to reduce the levels of both nitrate and phosphate.
Once the water has reached the Old Collegeville Road, a change can already be noticed in
the phosphate levels. The decrease in phosphate relates to the increase in nitrate. With
fewer phosphates present, there is less nutrients for algae. Less algae means that there
will be more nitrate left in the water. The sample analysis for this site supports this
theory.
It seems that most of the phosphate had already been removed from the river once
it reaches the area around Hw3. The nitrate levels, on the other hand, seem to be
influenced by sources other than the treatment plant. There are several farms and
Millstream Park near Hw 3. Both of these could have been treated with fertilizers or
other chemicals that would have run off the land during rainstorms and run into the water.
The Hw 50 site is located just below the headwaters of the Watab River, which is
the Big Watab Lake. There are several homes along the lake where the river starts, which
could cause fluctuations in the nitrate and phosphate levels. This site was found to have
relatively low concentrations of both nitrate and phosphate.
The site located on Hw 160 is nearby several farms and residential areas. Both of
these sources could have caused the slight increases noticed in both the nitrate and the
phosphate levels.
Millstream Park is the next site location. The park seems to have a well-groomed
lawn that is probably the result of much chemical treatment. Also, there are farms
located upstream that may dump into the river causing changes and increases in
phosphate concentrations.
The samples taken from the site at Hw2 may have increased levels of phosphates
due to all of the residential areas surrounding the site (this may also account for the
nitrate changes). The same holds true for the next site, which is located off of Hw4.
As the Watab River runs in to the Mississippi River, the phosphate level remains
about the same, while the nitrate level increases greatly. The Watab River flows through
the city of Sartell before it empties into the Mississippi, and this seems to be the probable
source for the nitrate increases.
In addition to testing water samples, the methods for analysis were also
researched and tested. In past summers, research has shown promising results using
instruments like the Nitrate Ion Selective Electrode. Results obtained from previous
weeks showed that most sites had rather low concentrations of nitrate initially. Based on
this, it was hypothesized that the nitrate electrode was not sensitive enough to get
accurate results (figure 2). After several attempts using the nitrate electrode, this method
was set aside and the research was continued by using the cadmium reduction column.
The results obtained for the phosphate concentrations through using the
ammonium molybdate solution and ascorbic acid also suggested that the method might
not be accurate enough when measuring low concentrations. Based on this concern an
alternative method using butyl acetate to extract the phospho-molybdate complex was
attempted. However, this method was found to have irreproducible results. This is
evident by the R2 values, which were about 0.6 on average. Because of repeated
difficulties in obtaining reproducible results, this method was also set aside and the
experiment was continued using the initial phosphate method using ascorbic acid and
ammonium molybdate (Figure 5).
CONCLUSION
The results from both the nitrate and phosphate analysis suggest that East Gemini
Lake and the SJU wetlands are both effective methods for the reduction of phosphate and
nitrate. Currently this method appears to be working. However, the concern for the
future is that the lake and wetlands will soon become saturated with nitrate and phosphate
and will no longer be able to reduce the levels and alternative methods will be needed.
FUTURE WORK
Potential avenues for future research involving the nitrate and phosphate levels
around SJU and St. Joseph include working towards developing new methods to reduce
the nitrate and phosphate levels (perhaps before the water even leaves the treatment
plant). Also, it would be of interest to collect samples from different areas of the
Mississippi River around where the Watab River joins the Mississippi. It would be
important to determine how much of an effect the water from the Watab River actually
has on the Mississippi. Another possible avenue for future research would be to find
more sensitive instruments and methods for analysis.
REFERENCES
1
Lasslo, Stephen et al. Phosphate Concentration Determination of the Watab
River Shed. Summer 2001.
2
Solomon et al. Biology. 5th ed. Pg 1012. 1985. Saunders College Publishing.
Orlando, FL.
3
Henderson, Sarah A. Comparison of Methods for the Analysis of Phosphate and
Nitrate in the Lakes of St. John’s University. May 22, 1998.
4
O’Neill, Marne. Analysis of Nitrate and Phosphate in the Lakes at St. John’s
University: A Study of Three Methods. May 10, 1999.
5
Wetzel, Robert G. Limnological analysis. 3rd ed. Pgs 88-97. New York:
Springer, c2000.
6
Moldestad, Eric. Method Determination for the Impact of the Saint John’s
Community on Nitrate Levels in the Watab River. Summer 2001.